摘要
本研究旨在制备抗登革病毒(dengue virus,DENV)血清型2 (DENV2)E蛋白Ⅲ区(envelope protein domainⅢ,EDⅢ)单克隆抗体(简称单抗)并研究其诊断特异性。合成编码DENV2-EDⅢ的基因并将其克隆入原核表达质粒pET21a;将重组质粒转入大肠埃希菌菌株BL21;用重组表达的EDⅢ免疫小鼠;将小鼠脾细胞与小鼠骨髓瘤细胞株SP2/0融合;将可产生识别抗EDⅢ单抗的杂交瘤细胞注入小鼠腹腔;采用蛋白G亲和层析法从腹水中纯化单抗;采用间接ELISA和间接免疫荧光法(indirect immunofluorescence assay, IFA)评估所制备的单抗识别DENV血清型1～4 (DENV1～4)的能力。本研究成功表达了DENV2-EDⅢ,并制备出3株杂交瘤细胞,杂交瘤细胞所分泌的小鼠源性单抗既可识别DENV2,又可交叉识别DENV1、DENV3和DENV4。
To develop monoclonal antibodies(mAb)specific for envelope protein domainⅢ(EDⅢ)of dengue virus serotype 2(DENV2)and test their diagnostic specificity,the gene encoding DENV2-EDⅢwas synthesized and cloned into prokaryotic plasmid pET21 a.The recombinant plasmid was transformed into competent Escherichia coli strain BL21.The recombinant DENV2-EDⅢwas used to immunize BALB/c mice.Splenocytes from immunized mice were fused with myeloma cell line SP2/0 and hybridomas were injected into the abdomen of BALB/c mice.Murine IgG was purified from the ascites and its ability to recognize DENV serotype 1 to 4(DENV1-4)was tested by indirect ELISA and indirect immunofluorescence assay.The results showed that DENV2-EDⅢwas successfully expressed and mAbs produced by hybridomas could recognize DENV2 as well as 3 other DENV serotypes(DENV1,DENV3 and DENV4)cross-reactively.
作者
段志良
周湧超
杨澜
陈新宇
文金生
金胜威
DUAN Zhi-liang;ZHOU Yong-chao;YANG Lan;CHEN Xin-yu;WEN Jin-sheng;JIN Sheng-wei(Department of Clinical Laboratory,the Second Affiliated Hospital of Wenzhou Medical University,Wenzhou 325027,China;Institute of Arbovirus,School of Basic Medical Sciences,WenzhouMedical University,Wenzhou325000,China;Department of Anesthesia and Critical Care,the SecondAffiliated Hospital of Wenzhou Medical University,Wenzhou325027,China)
出处
《现代免疫学》
CAS
CSCD
北大核心
2019年第2期120-125,共6页
Current Immunology
基金
浙江省自然科学基金(LY17C010004)
国家自然科学基金(81501363
31870159)
温州市科技计划项目(Y20150093)
关键词
登革病毒
E蛋白Ⅲ区
单克隆抗体
dengue virus
envelope protein domainⅢ
monoclonal antibody
作者简介
段志良(1976-),女,硕士生,副主任技师,主要从事虫媒病毒感染与宿主适应性免疫研究;通信作者:金胜威(E-mail:jinshengwei69@163.com)
